Structure elucidation of a novel oligosaccharide (Medalose) from camel milk

Article abstract of DOI:10.1016/j.molstruc.2017.10.006

Free oligosaccharides are the third most abundant solid component in milk after lactose and lipids. The study of milk oligosaccharides indicate that nutrients are not only benefits the infant's gut but also perform a number of other functions which include stimulation of growth, receptor analogues to inhibit binding of pathogens and substances that promote postnatal brain development. Surveys reveal that camel milk oligosaccharides possess varied biological activities that help in the treatment of diabetes, asthma, anaemia, piles and also a food supplement to milking mothers. In this research, camel milk was selected for its oligosaccharide contents, which was then processed by Kobata and Ginsburg method followed by the HPLC and CC techniques. Structure elucidation of isolated compound was done by the chemical degradation, chemical transformation and comparison of chemical shift of NMR data of natural and acetylated oligosaccharide structure reporter group theory, the ¹H, ¹³C NMR, 2D-NMR (COSY, TOCSY and HSQC) techniques, and mass spectrometry. The structure was elucidated as under: MEDALOSE[Figure presented]

Full text of DOI:10.1016/j.molstruc.2017.10.006

Accepted Manuscript
Structure elucidation of a novel oligosaccharide (Medalose) from camel milk
Lata Gangwar, Rinku Singh, Desh Deepak
PII:
S0022-2860(17)31337-6
10.1016/j.molstruc.2017.10.006
MOLSTR 24377
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 1 June 2017
Revised Date: 26 August 2017
Accepted Date: 2 October 2017
Please cite this article as: L. Gangwar, R. Singh, D. Deepak, Structure elucidation of a novel
oligosaccharide (Medalose) from camel milk, Journal of Molecular Structure (2017), doi: 10.1016/
j.molstruc.2017.10.006.
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ACCEPTED MANUSCRIPT
Structure Elucidation of a Novel Oligosaccharide (Medalose) from Camel
Milk
Lata Gangwar, Rinku Singh and Desh Deepak*
Department of Chemistry, University of Lucknow, Lucknow – 226007.
E-mail: deshdeepakraju@rediffmail.com
Abstract-
Free oligosaccharides are the third most abundant solid component in milk after lactose and
lipids. The study of milk oligosaccharides indicate that nutrients are not only benefits the
infant’s gut but also perform a number of other functions which include stimulation of
growth, receptor analogues to inhibit binding of pathogens and substances that promote
postnatal brain development. Surveys reveal that camel milk oligosaccharides possess varied
biological activities that help in the treatment of diabetes, asthma, anaemia, piles and also a
food supplement to milking mothers. In this research, camel milk was selected for its
oligosaccharide contents, which was then processed by Kobata and Ginsburg method
followed by the HPLC and CC techniques. Structure elucidation of isolated compound was
done by the chemical degradation, chemical transformation and comparison of chemical shift
of NMR data of natural and acetylated oligosaccharide structure reporter group theory, the
13
1H, C NMR, 2D-NMR (COSY, TOCSY and HSQC) techniques, and mass spectrometry.
The structure was elucidated as under:-
MEDALOSE
Key words- Milk Oligosaccharides, 2D NMR and Medalose.

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1. Introduction
Milk is a source of infant growth, development and contains proteins, fatty acids, minerals,
vitamins and carbohydrates especially, lactose and large number of oligosaccharides.
Oligosaccharides in milk exert various bioactivities and modulate the immune system [1].
Milk oligosaccharides participate in several protective and physiological roles including
immunoregulation and inhibition of pathogen adhesion in the gastrointestinal tract of infant.
Human milk oligosaccharides (HMOs) perform a number of functions including serving as
prebiotics to stimulate the growth of beneficial intestinal bacteria, as receptor analogues to
inhibit binding of pathogens, and as substances that promote postnatal brain development.
Human breast milk play a very important role in gut colonization and modulation of the
infants gut [2]. Sheep milk aggravates hiccup and dyspnoea. It eliminates pitta, kapha and fat.
It also contains fucose in its oligosaccharides which causes various biological activities [3].
Ancient literature of Ayurveda has declared the importance of cow milk as immuno-stimulant
and natural aphrodisiac, it improves intelligence, cures heart diseases and Leucoderma, also
cures bleeding disorders in intestine [4]. In Middle East and Mongolia camel milk is
consumed as a nutrient provider for infants’ development. Since the time of Chinggis Khaan
(Genghis Khan), Mongolians have used camel milk as a health-promoting product.
Mongolians literature says that fermented camel milk has been used for treatment of Edema
in pregnant women and as an antiscorbutic agent [5]. In India camel milk is used
therapeutically against dropsy, jaundice, problems of the spleen, tuberculosis, asthma,
anaemia, and piles [6]. Camel milk has proven to be beneficial for lung ailments and for the
treatment of tuberculosis [7]. Patients with chronic hepatitis had improved liver function after
being treated with camel milk [8].
In the present study we have described the structure elucidation of novel camel milk
oligosaccharide isolated from camel milk by the method of Kobata and ginsburg followed by
chemical degradation, chemical transformation and various one and two dimensional NMR
spectroscopy and Mass spectrometric analytical methods.

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2. Experimental-
2.1 General procedure
Same as described in our previous articles [9].
2.2 Isolation of camel milk oligosaccharides by the method of Kobata and Ginsburg-
Isolation of oligosaccharides from camel’s milk was done by the method of Kobata and
Ginsburg [10], which yielded 315gm of oligosaccharide mixture.
2.3 Acetylation of oligosaccharide mixture-
For acetylation 12 g of oligosaccharide mixture which gave positive phenol-sulphuric acid
test [11] was acetylated by standard method of acetylation by acetic anhydride and pyridine,
which yielded 13.5 gm acetylated mixture mixture of oligosaccharides [9].
2.4 Purification of compounds by Silica Gel Chromatography-
Separation or purification of acetylated oligosaccharides (13.5 gm) was carried over silica gel
(500gm) using varied proportion of Hex: CHCl3, CHCl3 and CHCl3: MeOH as eluents,
collecting fractions of 300ml each. All these fractions were checked on TLC and those
showing similar spots were taken together for further investigations. So ten fractions namely
I(4.28gm), II (736mg), III (3.29gm), IV (468mg), V (380mg) ,VI(2.007gm), VII(1.06gm),
VIII (767mg), IX (319mg) and X (137mg) were obtained. Substance B (95mg) was isolated
from fraction II by repeated column chromatography. The [α]D value of medalose acetate (B)
was +46.
2.5 Deacetylation of compounds Medalose Acetate
Medalose acetate (45mg) obtained from the column chromatography of acetylated
oligosaccharide mixture was dissolved individually in acetone (3 ml) and NH4OH (3.5ml)
was added and this was left overnight in a stoppered hydrolysis flask. After 24 h ammonia
was removed under reduced pressure and the compound was washed with (3 x 5 ml) CHCl3
(to remove acetamide) and the water layer was finally freeze dried giving the deacetylated
oligosaccharide i.e. Medalose (37mg).
Methylglycosidation/ Acid Hydrolysis of Medalose
Medalose (15mg) was ref1uxed with absolute MeOH (2 ml) at 70°C for 18 h in the presence
of cation exchange !R-l20 (H) resin. The reaction mixture was filtered while hot and filtrate
was concentrated. To a solution of methylglycoside of Medalose in 1,4-dioxane (I ml), 0.1 N
H2S04 (1 ml) was added and the solution was warmed for 30 minutes at 500C. The hydrolysis
was complete after 22 h. The hydrolysate was neutralized with freshly prepared BaCO3
filtered and concentrated under reduced pressure to afford α-and β-methylglucosides along

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with the GIc, Gal and GlcNAc. Their identification was confirmed by comparison with
authentic samples (TLC, PC).
2.6 Killiani Hydrolysis of Medalose
Medalose (15mg) was dissolved in 2 ml of Killiani mixture (AcOH-H2O-HCI, 7:11:2)[12]
separately and heated at 1000C for 1 h followed by evaporation under reduced pressure. It
was dissolved in 2 ml of H20 and extracted twice with 3 ml CHCl3. The aqueous residual
solution was made neutral by addition of 1-2 drops of 2N NaOH, to it and was evaporated
under reduced pressure to afford glucose, galactose and GlcNAc on comparison with
authentic samples of glucose, galactose and GlcNAc.
2.7 Description of Compound-
Compound Medalose-
Compound b Medalose C34H58N2O26 (37mg) viscous syrup, [α]D +52.80(c,4,H2O). Elemental
analysis: Calculated. %C 44.83, %H 6.37, %N 3.08 and found -%C 44.79, %H 6.29, %N
3.06. The molecular formula of compound Medalose was C34H58N2O26. For experimental
analysis, this compound was dried over P2O5 at 1000 C and 0.1 mm pressure for 8 hr.
The presence of sugar units in compound Medalose have been confirmed by NMR and Mass
spectrometry.
1H NMR values of Medalose in D2O:
2.00 ppm[s, 3H, NHCOCH3, β-GlcNAc (S-3)], 3.28 ppm [t, 1H, J=7.0 Hz, β-Glc(S-1), H-2],
3.93 ppm [, β-Gal (S-2), H-4],4.06 ppm [t, 1H, β-GlcNAc(S-3), H-3],4.47 ppm [d,2H, J=7.5
Hz, β-Gal (S-2 and S-4), H-1] ,4.53 ppm [d, 1H, J=7.8 Hz, β-GlcNAc (S-3), H-1],4.56 ppm
[d, 1H, J=9.0 Hz, β-GlcNAc (S-5), H-1], 4.67 ppm [d , 1H ,J=8.7 Hz, β-Glc (S-1), H-1], 5.23
ppm [d,1H, J=3.8 Hz, α-Glc (S-1),H-1].
13C NMR values of Medalose in D2O:
21.8 ppm [s,NHCOCH3]., 24.9 ppm [s,NHCOCH3], 91.8 ppm [α-Glc (S-1),C-1], 96.5 ppm
[β-Glc (S-1),C-1], 102.3 ppm [β-GlcNAc (S-3), C-1], 102.8 ppm [β-Gal (S-2 and S-4),& β-
GlcNAc (S-5), C-1], 173.6 ppm [s,NHCOCH3] .
1H NMR values of Acetylated Medalose in CDCl3:
4.48 ppm [d, 1H, J=8.4 Hz, β-GlcNAc (S-3), H-1], 4.49 ppm [d,2H, J=8.3 Hz, β-Gal (S-2 and
S-4), H-1], 4.51 ppm [d, 1H, J=9.0 Hz, β-GlcNAc (S-5), H-1], 5.68 ppm [d, 1H ,J=8.7 Hz, β-
Glc (S-1), H-1], 6.25 ppm [d,1H, J=3.6 Hz, α-Glc (S-1),H-1].
13C NMR values of Acetylated Medalose in CDCl3:

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89.1 ppm [α-Glc (S-1), C-1], 91.7 ppm [β-Glc (S-1),C-1], 101.1 ppm [β-GlcNAc (S-3& S-5),
C-1]., 101.3 ppm [β-Gal (S-2 & S-4), C-1]
ES Mass of Medalose:
933[M+Na]+, 910 [M+], 908 (95% in exp), 892, 861, 848(68% in exp), 844, 837, 819, 819,
789 (60 % in exp), 771, 758, 748 (33% in exp), 730, 703 (40% in exp), 699, 681 (20% in
exp), 659, 652 (10% in exp), 621, 611, 590, 579, 576, 558(5%), 545, 527, 496, 479,
465(100%), 406(18%), 402, 384(6%), 375, 357, 342, 325, 293, 275, 266, 260(28%), 239,
223(35%), 200, 190, 187(10%).
3. Result and Discussion-
Medalose, C34H58N2O26 [α]
+52.8 gave positive Phenol- sulphuric acid test[11], Feigl
D
test[13], Morgon-Elson test[14] showed the presence of normal and amino sugar(s) in the
compound. The HSQC spectrum of acetylated medalose acetate in CDCl3 showed the
presence of five cross peaks of six anomeric protons and carbons in the respective region at δ
6.25 ppm x 89.1 ppm (1C), δ 5.69 ppm x 91.7 ppm (1C), δ 4.48 ppm x 101.1 ppm (1C), δ
4.51 ppm x 101.1 ppm (1C) and δ 4.49 ppm x 101.3 ppm (2C) suggesting the presence of six
1
anomeric protons and carbons in it. Further H NMR spectrum of medalose acetate at 300
MHz in CDCl3 showed five signals for six anomeric proton at δ 6.25 ppm (1H), δ 5.69 ppm
(1H), δ 4.48 ppm (1H), δ 4.51 ppm (1H), and δ 4.49 ppm (2H) indicating that the Medalose
acetate may be pentasaccharide in its reducing form. It was further supported by appearance
of five signals for the six anomeric carbons at δ 89.1 ppm (1C), δ 91.7 ppm (1C), δ 101.1
13
ppm (1C), δ 101.1 ppm (1C) and δ 101.3 ppm (2C) in the C NMR spectrum of acetylated
1
Medalose in CDCl3. The H NMR spectrum of Medalose at 300 MHz in D2O exhibited five
doublets for six anomeric proton signals at δ 5.23 ppm (1H), 4.67 ppm (1H), 4.56 ppm (1H),
4.53 ppm (1H) and 4.47 ppm (2H) indicating that the compound may be a pentasaccharide in
its reducing form giving signals for α and β anomers of glucose at its reducing end.
Methylglycosidation of medalose by MeOH/H+ followed by its acid hydrolysis led to
isolation of α and β- methyl glucosides, which suggested the presence of glucose at the
reducing end of the oligosaccharide. It was also confirmed by the presence of two anomeric
proton signals at δ 5.23 ppm and δ 4.67 ppm for α and β-Glc. The pentasaccharide nature of
medalose was further confirmed by the presence of four doublets for six anomeric carbon at δ
13
91.8 ppm (1C), δ 96.5 ppm (1C),δ 102.3 ppm (1C) and δ 102.8 ppm (3C) in C NMR of
medalose in D2O. The five monosaccharides present in Medalose have been designated as S1,
S2, S3, S4, and S5 for convenience starting from reducing end. To confirm the monosaccharide

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constituents in Medalose, it was hydrolysed under strong acidic conditions. In Killiani
hydrolysis under strong acid condition, it gave three monosaccharides i.e. glucose, galactose
and N-acetyl-glucosamine, confirming that the pentasaccharide consists of three types of
1
monosaccharide units. The HNMR of Medalose acetate contains two anomeric proton
signals at δ6.25 ppm and δ5.69 ppm showed the presence of α and β-glucose (S1) present at
the reducing end. The anomeric proton present at δ5.69 ppm contain three cross peaks at
δ5.02 ppm, δ5.23 ppm and δ3.69 ppm in the TOCSY spectrum of Medalose acetate, out of
which the cross peak present at δ3.69 ppm was assigned for glycosidic linkage which was
later identified for H-4 of the reducing glucose by the COSY spectrum of Medalose acetate
confirming that H-4 of reducing glucose was available for glycosidic linkage by the next
monosaccharide. Further another anomeric proton doublet which was present at δ4.61 ppm
was due to presence of galactose which was linked to H-4 of reducing glucose, showing
presence of Lactosyl moiety at the reducing end in Medalose. Since the presence of glucose
was also confirmed at its reducing end of Medalose by methyl glycosidation/acid hydrolysis
was also supported by 1H NMR of Medalose which contains two anomeric proton signals for
α- and β-Glc at δ 5.23 ppm (J=3.8 Hz) and at δ 4.67 ppm (J=8.7 Hz)[15,16]. It also contains
another anomeric proton doublet at δ 4.47 ppm (J=7.5 Hz) which was due to presence of β-
Gal (S2) moiety [17] in the Medalose. The large coupling constant of anomeric proton signal
of Gal(S2) δ 4.47 ppm (J=7.5 Hz) confirmed the β glycosidic linkage between S2 and S1.
Presence of reducing glucose along with β-Gal moiety present in Medalose suggested the
presence of a lactosyl moiety i.e. Gal-β-(1→4) Glc in Medalose which was further confirmed
by β-Glc (S1) H-2 signal (a structure reporter group) [18] which appeared as a triplet at δ 3.29
ppm confirmed the presence of lactosyl moiety in Medalose. Further the anomeric proton of
β-galactose present at δ 4.48 ppm showed three cross peaks at δ5.15 ppm, 4.53 ppm and
δ3.73 ppm in the TOCSY spectrum of Medalose acetate, out of which the cross peak present
at δ3.73 ppm showed availability for glycosidic linkage by the next monosaccharide unit
which was later identified for H-6 of the β-galactose by the COSY spectrum of Medalose
acetate confirming that H-6 of β-galactose (S2) was available for glycosidic linkage by the
next monosaccharide unit. The presence of another anomeric proton signal present at δ4.51 in
1
the HNMR of Medalose acetate was due to presence of GlcNAc showed that the GlcNAc
was present as the third monosaccharide unit linked to Gal S2 at H-6. Further the presence of
another anomeric proton doublet at δ 4.53 ppm (J=7.8Hz) along with signal of amide methyl

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group at δ 2.00 ppm 1HNMR of Medalose in D2O, was due to the presence of β-GlcNAc (S3)
moiety [19]. The large coupling constant of anomeric proton signal of GlcNAc (S3) δ4.51
ppm (J=7.5 Hz) confirmed the β glycosidic linkage between S3 and S2. The H-4 proton
resonance of β Gal (S2), which appeared at δ 3.93 ppm implies that the β-GlcNAc (S3) may
be 1→6 linked to β-Gal (S2)(SRG)[20,21]. This was confirmed on the basis of presence of β-
Gal (S2), H-6 at δ3.84 ppm and C-6 at δ73.8 ppm and chemical shift analogies of β-GlcNAc
(1→6) Gal-β as given by Dua et.al (S3) [22]. Further the anomeric proton of β-GlcNAc
showed three cross peaks at δ4.12 ppm, δ3.62 ppm and δ3.80 ppm in TOCSY spectrum of
Medalose acetate. Further the COSY spectrum of Medalose acetate confirmed that signal at
chemical shift at δ4.12 ppm was due to H-2 methene proton of GlcNAc (S3), while the signal
at δ3.80 ppm and δ3.62 ppm was due to H-3 and H-4 protons of S3 respectively, out of which
the positions of H-3 and H-4 protons of β- GlcNAc (S3) at δ 3.80 ppm and δ 3.62 ppm
respectively which imply that H-3 and H-4 of β-GlcNAc (S3) were involved in the
glycosydation with the next monosaccharide unit. The presence of another anomeric proton
signal present at δ4.49 ppm was due to the presence galactose in the spectrum of Medalose
acetate. Since H-4 showed a signal at δ3.62 ppm confirmed that Gal S4 was glycosydically
linked to H-3 of S3. The large coupling constant of anomeric proton signal of Gal (S4) δ4.51
ppm (J=7.5 Hz) confirmed the β glycosidic linkage between S4 and S3. Further the presence
of another anomeric proton signal appeared at δ 4.47 ppm (J=7.5 Hz) which was due to
presence of a β-GlcNAc moiety (S5) confirmed that S5 (GlcNAc) was linked to H-4 of β-
GlcNAc (S3) in spectrum of Medalose in D2O. The large coupling constant of anomeric
proton signal of GlcNAc (S5) δ4.51 ppm (J=7.5 Hz) confirmed the β glycosidic linkage
between S5 and S3. The linkage between S3 and S4 was established on the basis of presence of
downfield shifted H-3 proton of GlcNAc (S3) at δ 4.06 ppm as triplet. It was further
confirmed by anomeric proton chemical shift value of this β-Gal moiety (S4), which was
identical with the anomeric proton chemical shift value of β-Gal (S2) of lactosyl moiety,
which is a structure reporter group for S4 and S3 linkage [23]. Another anomeric signals
appeared at δ 4.56 ppm (J=9.0 Hz) along with signal of methyl group at δ 2.00 ppm was due
to presence of β-GlcNAc moiety (S5) which was linked to H-4 of β-GlcNAc (S3).
1H and 13C NMR values in D2O (TABLE 1)

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1H NMR (in ppm)
13C NMR (in ppm)
Coupling Constt.(J)(in Hz)
5.23
4.67
4.47
4.53
4.47
4.56
91.8
96.5
3.8
8.7
7.5
7.8
7.5
9.0
α-Glc
β-Glc
102.8
102.3
102.8
102.8
β-Gal
β-GlcNAc
β-Gal
β-GlcNAc
The 13C NMR values of anomeric carbons and ring carbons of Medalose are given in table 2.
The various values of ring carbons are in accordance with 13C value of their respective
monosaccharides, which also supports the derived structure.
13C NMR values (in ppm) of Medalose (TABLE 2)
C-1
C-2
C-3
C-4
C-5
C-6
CO
CH3
91.8
73.6
74.9
70.9
60.9
71.0
61.9
71.65
75.2
73.5
78.5
72.4
74.9
78.01
77.59
70.7
70.9
74.9
70.8
74.9
76.18
75.29
60.44
62.0
73.8
62.0
60.9
62.0
α-Glc
96.5
β-Glc
102.8
102.3
102.8
102.8
β-Gal
78.01
70.9
173.6
173.6
21.8
24.9
β-GlcNAc
β-Gal
72.4
β-GlcNAc
The pentasaccharide nature of medalose was further confirmed by the spectral studies of
acetylated product of compound B. These studies are made on the basis of HOMOCOSY,
TOCSY and HSQC connectivities. The glycosidic linkages were assigned by the cross peaks
for glycosidically linked carbons with their protons in HSQC spectrum of medalose acetate.

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The values of these cross peaks are as- β-Glc (S1) H-4 and C-4 at δ3.79 ppm x 75.94 ppm
shows (1→4) linkage, β-Gal ( S2 & S4 ) H-6 and C-6 at δ 3.82 ppm x 73.67 ppm shows
(1→6) linkage, β-GlcNAc(S3) H-3 and C-3 at δ 3.88 ppm x 75.94 ppm shows (1→3) linkage
and also it’s H-4 and C-4 at δ 3.8 ppm 2 x 75.81 ppm shows (1→4) linkage respectively. The
ES MASS spectrum of medalose not only confirmed the derived structure but also confirmed
the sequences of the monosaccharides in medalose. The highest mass ion peak were recorded
m/z 933 which was due to [M+Na]+. It also showed the M+ 910 which confirmed that the
molecular weight of compound was 910. Further the mass fragments were formed by
repeated H transfer in the oligosaccharide and was accompanied by the elimination of
terminal sugar less water. The fragmentation pathway confirmed the sequence of
monosaccharide units in the pentasaccharide (scheme 1).

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CH OH
CH OH
2
O
S-5
CH OH
2
CH
2
2
O
HO
O
O
O
S-1
OH
S-2
OH
OH
S-3
O
OH
O
HO
NHCOCH
OH
3
OH
NHCOCH
3
CH OH
2
O
O
HO
m/z= 910
S-4
OH
H
OH
CH OH
2
CH OH
CH OH
CH
2
2
2
O
HO
O
O
S-5
OH
O
O
S-1
OH
S-3
OH
S-2
OH
OH
O
O
HO
NHCOCH
OH
3
OH
NHCOCH
3
H
m/z= 748 (I)
H
CH OH
2
CH OH
2
CH
2
O
HO
O
O
O
S-1
OH
S-3
OH
S-2
OH
OH
O
HO
OH
OH
NHCOCH
3
m/z=545 (II)
CH OH
OH
CH
2
2
O
O
HO
S-1
OH
S-2
OH
OH
O
OH
OH
H
m/z=342 (III)
CH OH
2
O
S-1
OH
OH
HO
OH
m/z=180 (IV)
SCHEME 1:- ES MASS FRAGMENTS OF COMPOUND MEDALOSE

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The pentasaccharide on fragmentation gave a mass ion peak at m/z 748(I), corresponding to
tetrasaccharide unit, which was due to loss of S-4 sugar unit i.e. Gal (S-4) sugar unit linked to
the S-3 of pentasaccharide. It was supported by its respective fragment at m/z 180, which
confirmed the presence of Gal (S-4) at non reducing end. The tetrasaccharide on
fragmentation gave a mass ion peak at m/z 545(II), which was due to loss S-5 sugar unit i.e.
GlcNAc (S-5) sugar unit linked to the S-3 of tetrasaccharide unit.. It was supported by m/z
527 (545-H2O). The trisaccharide on fragmentation gave mass ion peak at m/z 342(III),
which was due to loss of S-3 sugar unit i.e. GlcNAc (S-3) sugar unit linked to the S-2 of
trisaccharide unit. This disaccharide on further fragmentation gave a mass ion peak at m/z
180(IV), which was due to loss of S-2 sugar unit i.e. Gal (S-2) sugar unit linked to the S-1 of
disaccharide. The other mass fragments obtained at m/z892(910-H2O), m/z 844(892-
CH2OH), m/z771(844-CH2OH), m/z 848(910-2CH2OH), m/z 789(848-CH2CO-OH), m/z
758(789- CH2OH), m/z861(910- CH2OH-H2O), m/z 819(861- CH2CO), m/z 771(819-
CH2OH-OH), m/z837(910-CH2OH- CH2CO) and m/z819(837-H2O). The pentasaccharide
m/z 910 on fragmentation gave tetrasaccharide m/z748 (M-S4), which was further confirmed
by its other fragment ions at m/z 730(748-H2O), m/z 699(730-CH2OH), m/z 681(699-H2O),
m/z 621(681-CH2OHCHO), m/z 590(621-CH2OH), m/z 688(748-CH2OHCHO), m/z
652(688-2H2O), m/z 621(652-CH2OH), m/z 579(621-CH2CO), m/z 659(748-NHCOCH3-
CH2OH), m/z 611(659-CH2OH-OH), m/z 593(611-H2O), m/z 576(593-OH) and m/z
558(576-H2O). The tetrasaccharide m/z 748 on fragmentation gave trisaccharide m/z527 due
to (M-S5- H2O), which was further confirmed by its other fragment ions at m/z 465(527-
2CH2OH), m/z 406(465- CH2CO-OH), m/z 375(406- CH2OH), m/z 357(375- H2O), m/z
496(545- CH2OH- H2O), m/z479(496-CH2OH- H2O), m/z462(479-OH), m/z402(462-
CH2OHCHO), m/z 384(402- H2O) and m/z 342(384- CH2CO). The trisaccharide m/z 545 on
fragmentation gave disaccharide m/z342, which was further confirmed by its other fragment
ions at m/z 293(342- CH2OH- H2O), m/z 275(293- H2O), m/z 257(275- H2O), m/z 239(257-
H2O), m/z190(239- CH2OH- H2O), m/z 325(342-OH),m/z 266(325- CH2CO- OH), m/z
218(266- CH2CO- OH) and m/z 200(218- H2O).The disaccharide m/z 342 on fragmentation
gave monosaccharide m/z 180. Based on the results obtained from chemical degradation and
1
chemical transformation, mass spectrometry and H, 13C, HOMOCOSY, TOCSY, HSQC
NMR, the structure of the isolated pentasaccharide is deduced as

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OH
O
HO
HO
S-5
NHAc
OH
OH
HO
HO
OH
HO
HO
O
O
O
O
O
O
S-1
O
S-3
S-2
S-4
OH
O
O
H
OH
OH
NHAc
OH
MEDALOSE
4. Conclusion
From the above informations, we conclude that the structure of isolated camel milk
oligosaccharide, Medalose. This oligosaccharide was reported for the first time from any
1
natural source or any milk and elucidated with the help of spectroscopic technique like H,
13C, 2 DNMR (COSY, TOCSY and HSQC) spectroscopy and mass spectroscopy.
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Highlights
1. Isolation and structure elucidation of a novel oligosaccharide from camel milk.
2. Detailed structural analysis by 1H, 13C, 2D-NMR and Mass Spectrometry.
3. Traditional methods were also been used.
4. Biological activities of camel’s milk.